TRPM4 inhibition slows neuritogenesis progression of cortical neurons

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-024-01140-3.

is highly regulated by intracellular Ca 2+ (Ca 2 + i ) [4,5]; its absence arrests filopodia formation and neurite progression [6].Inhibitors of Ca 2 + i release or inhibitors of voltage gated calcium channels reduce neurite outgrowth [7], and complete suppression of Ca 2 + i causes growth cone loss and cessation of neuritogenesis in cerebellar neurons [8].Thus, ion channels and transporters affecting the resting membrane potential and intracellular calcium also regulate neurite elongation and neuronal development [9].
Calcium signals and membrane potential regulation are critical for neuritogenesis; changes in both can reduce migration and alter neurite number and development.TRPM4, a non-selective cation channel activated by Ca 2 + i and permeable to monovalent cations, modulates calcium levels by controlling the Ca 2+ driving force [10],

Introduction
Neuritogenesis is the process by which neurons undergo morphological changes leading to neurite formation, allowing them to acquire a polarized morphology and establish somatic, dendritic, and axonal compartments [1].This is the first step in neuronal development, particularly in excitatory cortical neurons, occurring alongside their migration from the ventricular zone to cortical areas [2].
Neurite formation begins with F-actin rearrangement, leading to membrane protrusion and lamellipodia and filopodia formation.During engorgement, microtubules advance into the lamellipodia and filopodia, leading to cylindrical neurite consolidation [3].This process by microtubule-tracking protein EB, participating in focal adhesion disassembly and migration [12,13].Studies indicate TRPM4 is critical for adhesion and migration of various non-neuronal cells, such as mastocytes, lymphocytes, and melanoma cell lines.TRPM4 is expressed in the prefrontal cortex at least from P0 [14], suggesting a potential role in development.However, its specific role in neuritogenesis has not been explored.This study assesses TRPM4 role in neuritogenesis and Ca 2 + i dynamics during early neurite development.

Results
To determine TRPM4 role in neuritogenesis, we assessed its expression in neurons during early development in culture.We performed immunostaining for TRPM4 and β-tubulin III in cortical neuron cultures at 1, 3, and 24 h after plating.TRPM4 was already expressed in neurons at 1 h after plating, with homogeneous expression in the soma.This pattern changed to somatic and neuritic localization at 3 h and became more pronounced at 24 h (Fig. 1A, B, and C).
To determine whether TRPM4 is coexpressed with F-actin at focal adhesions, a critical site for neurite outgrowth, we immunostained TRPM4 and used Phalloidin to detect F-actin.TRPM4 was coexpressed with F-actin, particularly in neurite growth cones (Fig. 1D), suggesting a role in neuritogenesis.
To examine the effect of TRPM4 inhibition on neuritogenesis, we used the model of polarity establishment with five stages: Stage 1, where spherical neurons start to extend filopodia and lamellipodia; Stage 2, where neurons present minor neurites; Stage 3, where one neurite grows to become an axon; and Stages 4 and 5, where neurons develop dendrites and form synapses, respectively [15].We inhibited TRPM4 with 9-Phenanthrol (10 and 30 µM) in cortical neurons right after plating.After 1, 3, or 24 h of treatment, we fixed the neurons, immunolabeled with β-tubulin III and Phalloidin, then imaged neuronal morphology using a confocal microscope, and classified the neurons in the neuritogenesis stages (Suppl.Methods).

Discussion
TRPM4 is involved in non-neuronal cell migration, forming part of focal adhesions and regulating cell movement.Gradients of Ca 2 + i are critical to promote neuritogenesis [6,16], particularly low levels of Ca 2 + i promote neurite outgrowth [6], in this context, our experiments shows that TRPM4 inhibition increases Ca 2 + i , thus may produce an opposite effect, and altering the optimal level of Ca 2 + i necessary for neuritogenesis.This Ca 2 + i increase may be due to the membrane potential of the neuron, which at this early stage is more depolarized and less responsive, since TRPM4 is expressed at least from DIV0 [14], TRPM4 could play critical roles in controlling depolarization and the Ca 2 + i influx through VGCC during initial stages of development and impacting neurite outgrowth.
(See figure on previous page.)Fig. 1 TRPM4 inhibition reduces neurite development progression of cortical neurons.In (A) Immunostaining with TRPM4 and β-tubulin III at 1 h and (B) 3 h and (C) 24 h post disaggregation.(D) Zoom of the (C) showing the Immunostaining of TRPM4 and F-actin at 24 h, the white box indicates the expression in the growth cone.Scale bar = 10 μm.(E) Summary graph showing the effect of TRPM4 inhibition with 10 µM 9-Ph and 30 µM 9-Ph in the percentage of neurons in stage 1 and 2 of the neuritogenesis after 1 h (N = 4).(F) Summary graph showing the effect of TRPM4 inhibition with 10 µM 9-Ph and 30 µM 9-Ph in the percentage of neurons in stage 1 and 2 of the neuritogenesis after 3 h (N = 4).(G) Summary graph showing the effect of TRPM4 inhibition with 10 µM 9-Ph in the percentage of neurons in stage 1, 2 and 3 of the neuritogenesis after 24 h post culture (N = 2).Summary graph showing the length (H) and number (I) of the primary neurite before and after 10 µM 9-Ph.(J) Shows the primary neurite length in neurons transfected with shRNA-TRPM4 and the scramble sequence.Summary graph showing the quantification of the (K) maximum fluorescence and the (L) decay time of the signal induced by 10 µM Cch and 10 µM Cch + 10 µM CBA (N = 3).(M) Representative graph showing the Ca 2 + i response, gray lines showing all neurons recorded, red line shows the average of the signal.Data is shown as the mean ± 95 C.I. and the right plot shows the mean difference, the p-value is shown above the graph TRPM4 is expressed in lamellipodia where the presence of F-actin is critical for cell migration, our results shows that TRPM4 is coexpressed in areas rich in F-actin in the growth cone, thus in this area TRPM4 may participate in the tight control of the Ca 2 + i which is critical for neurite outgrowth [6].In non-excitable cells, TRPM4 participates in the focal adhesion turnover by modulating EB proteins, reducing focal adhesion disassembly, and decreasing lamellipodia extension [12,13]; a similar effect may be playing TRPM4 in neurite outgrowth.We found that inhibiting TRPM4 with 9-Ph at the beginning of neuritogenesis is relevant for neurite stage progression.However, after 24 h inhibition, we observed no significant change in neurite length or number.Interestingly, TRPM4 silencing for 48 h significantly reduce neurite length, suggesting that TRPM4 also participates in later stage of neuritogenesis.In this context the use of strategies to silence the channel at earlier stages are necessary to complement our pharmacological approach and to fully investigate the effects of TRPM4 on the initial developmental stages of neuritogenesis.In conclusion, our findings suggest that TRPM4 plays a significant role in neuritogenesis by modulating intracellular calcium.